The purpose of this project is to compare thermodynamic data obtained in vitro, for the purine salvage reactions to the measured steady-state levels of purine and pyrimiding nucleobases, nucleosides, nucleotides and related co-factor compounds, in vivo. The hypothesis being that the purine salvage enzyme catalyze reactions that approach near-equilibrium, in vivo, and thus that rates of synthesis of the necessary co-factors such as ribose 1-phosphate (R1-P), phosphoribosylpyrophosphate (PRPP), inorganic phosphate (Pi), and inorganic pyrophosphate (PPi), could have profound influence on the relative amounts of free bases and nucleosides that would be available for nucleotide and urate production. Current methods for the extraction and quantitation of purine and pyrimidine nucleobases and nucleosides as well as for R1-P and PRPP are suspect and have led to confusion and possibly misinterpretation of scientific data. Therefore, we have begun this project by attempting to improve on current methodologies for measuring the aforementioned metabolites in liver extracts. A high performance liquid chromatographic procedure has been developed for measuring purine and pyrimidine nucleobases and nucleosides in rat hepatocytes that we believe is superior to any such method described thus far. Progress is also being made on improving techniques for the extraction and quantitation of R1-P and PRPP. The apparent equilibrium constants for the purine salvage reactions catalyzed by purine nucleoside phosphorylase has been completed as well as for the ancillary reactions; phosphoribomutase and 5' nucleotidase. The equilibrium constant for the phosphoribosyltransferases will be completed once PRPP can be measured. Once these in vivo measurements are made it can be determined whether or not the salvage reactions approach near-equilibrium, in vivo, and, if so, it will be possible to assess the influence fluctuations in the magnitude of the co-factor couples; PRPP/PPi and R1-P/Pi, have on the rates of nucleotide, nucleic acid and uric acid synthesis in a variety of cell types.